Estrogenic extracts of Astragalus membranaceus fisch. bge. var. mongolicus bge. of the Leguminosae family and uses thereof

ABSTRACT

Estrogenic extracts of  Astragalus membranaceus  Fisch. Bge. Var.  mongolicus  Bge. of the Leguminosae Family are provided. Also provided are methods of using said extracts to achieve an estrogenic effect, especially in a human, e.g. a female human. In some embodiments, the methods include treatment of climacteric symptoms. In some embodiments, the methods include treatment of estrogen receptor positive cancer, such as estrogen responsive breast cancer. In some embodiments, the methods include treatment or prevention of osteoporosis.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/970,806, filed Sep. 7, 2007; and is a division ofU.S. Nonprovisional patent application Ser. No. 12/205,686, filed Sep.5, 2008 now abandoned, the contents of each are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to plant extract compositions, and moreparticularly to compositions comprising extracts of plant speciesbelonging to the species Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae family. The invention further relatesto methods of using and methods of making such plant extractcompositions.

BACKGROUND OF THE INVENTION

Hormone replacement therapy (HRT) has been used successfully to treat avariety of conditions, such as osteoporosis, increased risk ofcardiovascular disease in post-menopausal women and climactericsymptoms, such as hot flashes, decreased libido and depression. However,HRT with estradiol (E₂), either alone or in combination with progestin,can lead to undesirable effects. In fact, a recent Women's HealthInitiative (WHI) study was abruptly halted when preliminary resultsshowed that HRT was associated with a 35% increased risk of breastcancer.

Breast cancer can be treated or prevented by using a so-called selectiveestrogen receptor modulator (SERM), such as tamoxifen. (Before theapproval of tamoxifen, breast cancer treatment of pre-menopausal womenoften included removing the ovaries in order to reduce thecancer-stimulating effect of estrogen.) Tamoxifen appears to selectivelyblock the cancer-inducing effects of estrogen in breast tissues ofpre-menopausal women. Another SERM, raloxifene, has been approved fortreatment of osteoporosis as an alternative to estrogen replacement. Inaddition to selectively inducing estrogenic effects in bone tissue,long-term administration of raloxifene was also shown to be associatedwith reduction in the rate of breast cancer in the Multiple Outcomes ofRaloxifene Evaluation (MORE) study.

While SERMs such as tamoxifen and raloxifene provide selective reductionin estrogen's cancer-inducing effects in the breast, they are notwithout their risks. For example both tamoxifen and raloxifene therapyhave been associated with increased incidence of hot flushes, andtamoxifen therapy has been shown to increase the risk of uterine(endometrial) cancer.

Despite the success of estrogen replacement therapy in treatingosteoporosis, coronary heart disease and climacteric symptoms, and ofSERMs like tamoxifen and raloxifene in treating breast cancer andosteoporosis, there remains a need for compositions having estrogenicproperties. Additionally, given the increasing cost of producing drugcompounds, there is a need for additional estrogenic compositions thatmay be obtained from natural sources.

Various cultivars of Astragalus membranaceus Fisch. Bge. Var. mongolicusBge. of the Leguminosae family is grown in northern and eastern China.Various other varieties grow all over the world. The root is collectedin the spring and autumn. It is washed clean, fibrous roots are removed.It is than sun dried.

There is no known report of using extracts of Astragalus membranaceusFisch. Bge. Var. mongolicus Bge. of the Leguminosae family as estrogeniccompositions.

There is a need for estrogenic compositions that are readily obtainedfrom natural sources. There is also a need for methods of making suchcompositions. There is also a need for methods of using such estrogeniccompositions.

SUMMARY OF THE INVENTION

The present inventor has identified a need for estrogenic compositionsuseful for the treatment of one or more disease states associated withthe estrogen receptor. The inventor has also identified a need forestrogenic compositions that do not increase the risk or likelihood thata patient administered the compositions will suffer from another diseasestate associated with an estrogen receptor. The inventor has likewiserecognized a need for an estrogenic composition that will reduce therisk of one or more estrogen receptor mediated disease states while, atthe same time, treating another estrogen receptor mediated diseasestate. The inventor has also identified a need for estrogeniccompositions that are readily obtained from natural sources, as well asa need for methods of making and using such estrogenic compositions. Thedisclosure herein meets such needs and provides related advantages aswell.

The invention provides a plant extract composition that contains anextract of a plant species of the species Astragalus membranaceus Fisch.Bge. Var. mongolicus Bge. of the Leguminosae Family. Some embodimentsprovide an extract of a plant species selected from the taxonomicspecies Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family. In some embodiments, the extract is either anaqueous extract, an ethanolic extract, a purified extract or apartitioned extract. In some embodiments, the extract is an ethanolicextract. Some embodiments provide a composition that contains an extractof a plant species of the species Astragalus membranaceus Fisch. Bge.Var. mongolicus Bge. of the Leguminosae Family for use in themanufacture of a medicament. In some embodiments, the medicamentpossesses an estrogenic effect. In some embodiments, the estrogeniceffect is at least one effect selected from the group consisting of:treating or preventing at least one climacteric symptom; treating orpreventing osteoporosis; treating or preventing uterine cancer; andtreating or preventing cardiovascular disease. In some embodiments, theestrogenic effect includes treating or preventing at least oneclimacteric symptom selected from the group consisting of treating orpreventing hot flashes, insomnia, vaginal dryness, decreased libido,urinary incontinence and depression. In some embodiments, the estrogeniceffect includes treating or preventing osteoporosis. In someembodiments, the estrogenic effect includes treating or preventing hotflashes. In some embodiments, the estrogenic effect includes treating orpreventing uterine cancer or breast cancer. In some embodiments, theestrogenic effect does not include increasing the risk of mammaryhyperplasia, mammary tumor, uterine hyperplasia, uterine tumor, cervicalhyperplasia, cervical tumor, ovarian hyperplasia, ovarian tumor,fallopian tube hyperplasia or fallopian tube tumor. In some embodiments,the estrogenic effect includes decreasing the risk of mammaryhyperplasia, mammary tumor, uterine hyperplasia, uterine tumor, cervicalhyperplasia, cervical tumor, ovarian hyperplasia, ovarian tumor,fallopian tube hyperplasia or fallopian tube tumor.

Some embodiments provide the use of the plant extract composition of forthe preparation of a medicament. In some embodiments, the medicamentpossesses an estrogenic effect. In some embodiments, the estrogeniceffect is at least one effect selected from the group consisting of:treating or preventing at least one climacteric symptom; treating orpreventing osteoporosis; treating or preventing uterine cancer; andtreating or preventing cardiovascular disease. In some embodiments, theestrogenic effect includes treating or preventing at least oneclimacteric symptom selected from the group consisting of: hot flashes,insomnia, vaginal dryness, decreased libido, urinary incontinence,headache and depression. In some embodiments, the estrogenic effectincludes treating or preventing osteoporosis. In some embodiments, theestrogenic effect includes treating or preventing hot flashes. In someembodiments, the estrogenic effect includes treating or preventinguterine cancer or breast cancer. In some embodiments, the medicamentcauses no statistically significant increase in risk of mammaryhyperplasia, mammary tumor, uterine hyperplasia, uterine tumor, cervicalhyperplasia, cervical tumor, ovarian hyperplasia, ovarian tumor,fallopian tube hyperplasia, fallopian tube tumor. In some embodiments,the medicament causes a decrease in the risk of mammary hyperplasia,mammary tumor, uterine hyperplasia, uterine tumor, cervical hyperplasia,cervical tumor, ovarian hyperplasia, ovarian tumor, fallopian tubehyperplasia, fallopian tube tumor.

Some embodiments provide a method of eliciting an estrogenic effect,comprising administering to a subject an estrogenically effective amountof the plant extract composition. In some embodiments, the extract iseither an aqueous extract, an ethanolic extract, a purified extract or apartitioned extract. In some embodiments, the extract is an ethanolicextract. In some embodiments, the estrogenic effect is at least oneeffect selected from the group consisting of: treating or preventing atleast one climacteric symptom; treating or preventing osteoporosis;treating or preventing uterine cancer; and treating or preventingcardiovascular disease. In some embodiments, the estrogenic effectincludes treating or preventing at least one climacteric symptomselected from the group consisting of treating or preventing hotflashes, insomnia, vaginal dryness, decreased libido, urinaryincontinence and depression. In some embodiments, the estrogenic effectincludes treating or preventing osteoporosis. In some embodiments, theestrogenic effect includes treating or preventing hot flashes. In someembodiments, the estrogenic effect includes treating or preventinguterine cancer. In some embodiments, the estrogenic: effect does notinclude increasing the risk of mammary hyperplasia, mammary tumor,uterine hyperplasia, uterine tumor, cervical hyperplasia, cervicaltumor, ovarian hyperplasia, ovarian tumor, fallopian tube hyperplasia,fallopian tube tumor. In some embodiments, the estrogenic effectincludes decreasing the risk of mammary hyperplasia, mammary tumor,uterine hyperplasia, uterine tumor, cervical hyperplasia, cervicaltumor, ovarian hyperplasia, ovarian tumor, fallopian tube hyperplasia,fallopian tube tumor.

Some embodiments disclosed herein provide a method of activating a geneunder control of an estrogen response element, comprising administeringto a cell having an estrogen response element operatively linked to thegene and an estrogen receptor an amount of the plant extract compositionsufficient to activate said gene. In some embodiments, the cell is invitro. In some embodiments, the cell is in vivo. In some embodiments,the cell is in an ERα+ breast tissue. In some embodiments, the cell isin an ERβ+ breast tissue. In some embodiments, the cell is in anERα/ERβ+ breast tissue. In some embodiments, the estrogen responseelement is expressed in a transformed cell. In some embodiments, theestrogen response element and the estrogen receptor are expressed in thecell. In some embodiments, the estrogen response element isheterologously expressed in the cell. In some embodiments, the estrogenresponse element and the estrogen receptor are heterologously expressedin the cell. In some embodiments, the cell is selected from the groupconsisting of a U937, a U205, a MDA-MB-435 and a MCF-7 cell transformedwith an ERE-controlled gene. In some embodiments, the cell expressesERα. In some embodiments, the cell expresses ERβ. In some embodiments,the ERE-controlled gene is ERE-tk-Luc.

Some embodiments provided herein provide a method of repressingexpression of a TNF RE-controlled gene, comprising administering to acell comprising a gene under control of a TNF response element and anestrogen receptor in an amount of the plant extract compositioneffective to repress said TNF RE-controlled gene. In some embodiments,the TIME RE-controlled gene is TNF-α. In some embodiments, the TNFRE-controlled gene is TNF RE-Luc. In some embodiments, the cell is invitro. In some embodiments, the cell is in vivo. In some embodiments,the cell is in an ER+ breast tissue. In some embodiments, the cell is inan ERα breast tissue. In some embodiments, the cell is in an ERβ+ breasttissue. In some embodiments, the TNF response element is endogenouslyexpressed. In the cell. In some embodiments, the TNF response elementand the estrogen receptor are endogenously expressed in the cell. Insome embodiments, the TNF response element is heterologously expressedin the cell. In some embodiments, the TNF response element and theestrogen receptor are heterologously expressed in the cell. In someembodiments, the cell contains an estrogen receptor gene, is transformedwith an TNF response element-controlled gene, and is selected from thegroup consisting of a U937, a U205, a MDA-MB-435 and a MCF-7 cell. Insome embodiments, the estrogen receptor gene is a gene expressing ERα.In some embodiments, the estrogen receptor gene is a gene expressingERβ.

Some embodiments provide a method of making a plant extract, comprisingobtaining a quantity of plant matter from a plant of the speciesAstragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLegurninosae family and contacting said plant matter with an extractionmedium comprising water, ethanol or both at a temperature between about25° C. and 100° C. and separating said extraction medium from saidplant. In some embodiments, the temperature is between about 50° C. and80° C. In some embodiments, the temperature is about 75° C.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graph of luciferase expression in U937 (human monocytes)cells transformed with DNA encoding estrogen response element linked tothe minimal thymidine kinase (tk) promoter and a sequence encodingluciferase (Luc) in response to varying concentrations of estradiol (E₂)in the presence of either estrogen receptor alpha (ERα), estrogenreceptor beta (ERβ) or both. ERβ has much less stimulatory effect on theERE than does ERα in the presence of E₂.

FIG. 2 is a graph of luciferase expression in MDA-MB-435 (humanmetastatic breast cancer) cells transformed with DNA encoding estrogenresponse element linked to the minimal thymidine kinase (tk) promoterand a sequence encoding luciferase (Luc) in response to varyingconcentrations of estradiol (E₂) in the presence of either estrogenreceptor alpha (ERα), estrogen receptor beta (ERβ) or both. ERβ has muchless stimulatory effect on the ERE than does ERα in the presence of E₂.Remarkably, when ERα and ERβ are coexpressed in this cell line, ERβexpression greatly reduces the ERE stimulatory effect of ERα in thepresence of E₂.

FIG. 3 is a graph of luciferase expression in U2OS (human osteosarcoma)cells transformed with DNA encoding estrogen response element linked tothe minimal thymidine kinase (tk) promoter and a sequence encodingluciferase (Luc) in response to varying concentrations Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familyof the Liliaceae family of the Oleaceae Family in the presence of eitherestrogen receptor alpha (ERα) or estrogen receptor beta (ERβ).Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae family of the Liliaceae family of the Oleaceae Family hasstimulatory effect on the ERE with ERβ, and it has none with ERα.

FIG. 4 is a graph of luciferase expression in U2OS (human osteosarcoma)cells transformed with DNA encoding tumor necrosis factor responseelement (TNF RE) linked to the minimal thymidine kinase (tk) promoterand a sequence encoding luciferase (Luc) in response to varyingconcentrations of Astragalus membranaceus Fisch. Bge. Var. mongolicusBge. of the Leguminosae family of the Liliaceae family of the OleaceaeFamily in the presence of either estrogen receptor alpha (ERα) orestrogen receptor beta (ERβ). Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae family of the Liliaceae family of theOleaceae Family shows repression of TNF-RE with ERβ but not with ERα.

FIG. 5 is a graph of Estrogen receptor binding assays. Estrogen receptorbinding assays were carried out in black Corning low volume 384-wellplates according to the protocol outlined in the datasheet that wasincluded with each kit (Invitrogen #P2698 or #P2700). The total volumeper each well was 20 μL and all reactions were performed in triplicate.Compounds were diluted to a 2× concentration in the 1× Screening bufferprovided. Final concentrations of compounds ranged from 1.4×10⁻² mg/mLto 2.724×10⁻¹¹ mg/mL. Each well consisted of 10 μL of the 2×concentration of the compound and 10 μL of a 2× concentration of theFluoromone and estrogen receptor that was also provided with each kit.Once the diluted compounds were mixed with the fluoromone and estrogenreceptor, the plate was gently mixed and incubated for 2 hours in thedark. Each plate was read using a fluorescent polarization reader (TecanGeniosPro) and the data was analyzed using Sigmaplot. Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familyof the Liliaceae family of the Oleaceae Family has similar bindingaffinity in the presence of either estrogen receptor alpha (ERα),estrogen receptor beta (ERβ).

FIG. 6 is a graph of luciferase expression in U2OS (human osteosarcoma)cells transformed with DNA encoding estrogen response element linked tothe minimal thymidine kinase (tk) promoter and a sequence encodingluciferase (Luc) in response to varying concentrations Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familyof the Liliaceae family of the Oleaceae Family in the presence ofestrogen receptor beta (ERβ) in competition to the known SERMantagonists raloxifene and tamoxifen. Astragalus membranaceus Fisch.Bge. Var. mongolicus Bge. of the Leguminosae family of the Liliaceaefamily of the Oleaceae Family stimulatory effect on the ERE with ERβ isinhibited in the presence of tamoxifen and raloxifene.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention provides a plant extract composition that contains anextract of the taxonomic species of plant referred to as Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae Family.The invention also provides estrogenic methods of using the inventivecompositions. Such estrogenic methods include in vivo methods and invitro methods. The estrogenic compositions possess the ability toantagonize the activation of a gene under control of the estrogenresponse element (ERE) by estradiol (E₂) and an estrogen receptor (ER).Accordingly, suitable in vivo methods include treatment and/orprevention of medical indications that are responsive to antagonism ofE₂-stimulated activation of gene expression. Suitable in vitro methodsinclude use in methods of activating a gene under control of theestrogen response element (ERE) and methods of repressing expression ofa gene under control of the tumor necrosis factor response element (TNFRE). The invention further provides methods of making the inventiveextracts.

Breast neoplasms are the most common cancers diagnosed in women. In2000, 184,000 new cases of breast cancer were diagnosed and 45,000 womendied from breast cancer. Although the cause of breast cancer is probablymultifactorial, there is compelling clinical, epidemiological andbiological research that indicate estrogens promote breast cancer: (a)Hormone replacement therapy (HRT) is associated with a 35% increasedrisk of breast cancer by a meta-analysis of 51 studies; (b) Breastcancer can be prevented with tamoxifen or raloxifene, which bind to ERsand antagonize the actions of estrogens in breast cells; (c) Bilateraloophorectomy in premenopausal women with breast cancer leads toincreased survival; (d) Greater exposure to estrogens (early menarche orlate menopause, relative risk=1.3 and 1.5 to 2.0, respectively)increases the incidence of breast cancer; (e) Estrogens increase theproliferation of ER positive breast cancer cells; and (f) Estrogensincrease the production of growth promoting genes, such as cyclin Dl,c-myc, and c-fos.

Approximately 60-70% of breast tumors contain estrogen receptors. Forseveral decades, breast tumors have been analyzed for the presence ofERs. Approximately 70% of ER+ tumors are responsive to antiestrogentherapy. This observation has led to the notion that ER+ tumors have abetter prognosis than ER negative tumors. However, the discovery of ERβhas complicated these interpretations and has raised some profoundclinical questions. Understanding the role of ERα and ERβ is ofparamount importance, because the current methods of determining whethertumors are ER+ uses an antibody that only detects ERα. Thus, moststudies examining the effects ERs in breast tumors on clinical outcomesreflect the only ERα status. However, several recent studies havedetected the presence of ERβ mRNA in human breast tumors. Most of thestudies relied on RT-PCR to measure ERβ, because of the lack of specificand sensitive antibodies to ERβ. Dotzlaw et al. were the first to detectERβ in breast tumor biopsies by RT-PCR. They found 70% of the breasttumors expressed ERβ and 90% expressed ERα. Furthermore, theydemonstrated that several ER negative cell lines also express ERβ mRNA.These findings suggest that ERβ is highly expressed in breast tumors,and that both ERα and ERβ are often coexpressed in many tumors. In fact,some ER—tumors contain ERβ. Dotzlaw et al. also showed that ERβ mRNA issignificantly lower in ER+/PR− (PR being progestin receptor) tumorscompared to ER+/PR+ tumors. The authors suggested that this observationindicates that ERβ expression is associated with a poorer prognosis,because ER+/PR+ are more likely to respond to tamoxifen. Other studiessuggest that the presence of ERβ confers a poor prognosis. Speirs et al.found that most breast tumors express ERβ mRNA alone or in combinationwith ERα mRNA. Those tumors that express both ERα and ERβ mRNA wereassociated with positive lymph nodes and tended to be characterized ashigher grade tumors. Furthermore, increased ERβ expression occurs inMCF-10F cells treated with chemical carcinogens, suggesting that theexpression of ERβ may contribute to the initiation and progression ofbreast cancer. Recently, Jensen et al. analyzed the expression of ERβ in29 invasive breast tumors by immunohistochemistry (IHC). They found thatERβ expression was associated with an elevation of specific markers ofcell proliferation, Ki67 and cyclin A. Moreover, the highest expressionof these proliferation markers was present in ERα+/ERβ+ tumors. Althoughthe number of ERα-TERβ+ cases were very small (n=7) the authorssuggested that ERβ mediates cell proliferation in breast tumors. Speirset al. also reported ERβ mRNA is significantly elevated in thetamoxifen-resistant tumors compared to tamoxifen-sensitive tumors.

In contrast, other studies indicate that the presence of ERβ confers afavorable prognosis. Iwao et al. demonstrated that ERα mRNA isup-regulated and ERβ mRNA is down-regulated as breast tumors progressfrom preinvasive to invasive tumors. Using IHC of frozen tumor sectionsJarvinen et al. found that ERβ expression was associated with negativeaxillary node status, low grade, and low S-phase fraction. A study byOmoto et al. also found that ERβ positive tumors correlated with abetter prognosis than ERβ negative tumors, because the disease-freesurvival rate was higher in tumors containing ERβ. ERβ expression alsoshowed a strong association with the presence of progesterone receptorsand well-differentiated breast tumors. It has also been reported thatthe levels of ERβ are highest in normal mammary tissue and that itdecreases as tumors progress from pre-cancerous to cancerous lesions.These studies indicate that ERβ may function as a tumor suppressor andthat the loss of ERβ promotes breast carcinogenesis. In a study by Mannet al. it was shown that the expression of ERβ in more than 10% ofcancer cells was associated with better survival in women treated withtamoxifen. The aggregate of these studies indicates the presence of ERβconfers a favorable prognosis. Consistent with RT-PCR and IHC data is areport that showed that adenovirus-mediated expression of ERβ resultedin a ligand-independent inhibition of proliferation of the ER negativecell line, MDA-MB-231.

These results demonstrate that the role of ERβ in the pathogenesis andprognosis of breast cancer is unclear. Several reasons may explain theapparent discrepancy among these studies. First, there may be a poorcorrelation between ERβ mRNA and ERβ protein. This notion is consistentwith the presence of ERβ mRNA in some ER negative cell lines that do nothave detectable ERs by ligand binding assays. Second, the IHC studiesused different commercially available ERβ antibodies that have beenpoorly characterized for specificity and sensitivity. Third, most of theconclusions have been based on a few breast cancer cases. Clearly, morestudies are needed to clarify the role of ERα and ERβ in breast cancer.

Role of SERMs as adjuvant therapy and chemoprevention in breast cancer:Because estrogens promote the proliferation of breast cancer cells,several therapeutic approaches have been implemented to block thiseffect of estrogens on breast tumors. These strategies, includingovarian ablation, antiestrogens, gonadotropin releasing hormone analogsor aromatase inhibitors, work by either decreasing the production ofestrogens or blocking the action of estrogens. All of these strategiesnon-selectively block the action of both ERα and ERβ. The most commonapproach used clinically to prevent and treat breast tumors are theselective estrogen receptor modulators (SERMs), tamoxifen andraloxifene.

Tamoxifen is a non-steroidal triphenylethylene derivative that is theprototype SERM, because it exhibits antagonistic action in some tissues,such as the breast, but has agonist actions in other tissues such as theendometrium and bone. Tamoxifen has been extensively studied for itsclinical effectiveness as an adjuvant therapy to reduce the recurrencesof breast tumors in women with estrogen receptor-positive breast cancer.Five years of tamoxifen therapy reduces the risk of recurrences by 42%,mortality from breast cancer by 22% and a second contralateral primarybreast tumor. Approximately, ⅔ of ER positive breast tumors respond totamoxifen, whereas very little evidence indicates that women with ERnegative tumors benefit from adjuvant tamoxifen. Most recently, the U.S.Breast Cancer Prevention Trial (BCPT) demonstrated that tamoxifenreduces the risk of primary invasive breast cancer by 49% in womenconsidered to be at high risk for breast cancer. These studiesdemonstrate that tamoxifen is a first-line effective adjuvant therapy inwomen with a history of breast cancer and is an effectivechemoprevention agent for women who are high risk for developing breastcancer.

Raloxifene is a member of the benzothiophene class of SERMs that hasrecently been approved for the prevention and treatment of osteoporosis.Raloxifene has not been evaluated for effectiveness as an adjuvanttherapy for women with breast cancer. However, the Multiple Outcomes ofRaloxifene (MORE) trial evaluated the effect of raloxifene on preventingbreast cancer. The MORE trial was a randomized, placebo-controlledthree-year study of 7705 postmenopausal women who have osteoporosis. Inthe MORE trial, 13 cases of breast cancer were found among the 5129women in the raloxifene treatment group versus 27 among the 2576 womenwho received placebo (RR=0.24) after a median follow-up of 40 months.Like tamoxifen, raloxifene is effective at reducing the incidence ofestrogen receptor positive tumors, but not estrogen receptor negativetumors. Additional evidence for a role of estrogens in promoting breastcancer comes from a recent study that showed raloxifene only preventsbreast cancer in postmenopausal women that have detectable levels ofserum estradiol.

Structure of Estrogens Receptors: The fact that SERMs only work on ERpositive tumors indicates that they need to interact with estrogenreceptors in order to exert its protective effects on the breast. Thereare two known estrogen receptors, ERα and ERβ, which are members of thesteroid nuclear receptor superfamily. ERα was first cloned in 1986, andsurprisingly about 10 years later a second ER was discovered, termedERβ. ERα contains 595 amino acids, whereas ERβ contains 530 amino acids.Both receptors are modular proteins made up of three distinct domains.The amino-terminus domain (A/B domain) is the least conserved region,exhibiting only a 15% homology between ERα and ERβ. This domain harborsan activation function (AF-1) that can activate gene transcriptionactivation in the absence of estradiol. The central region of ERscontains two zinc finger motifs that bind to an inverted palindromicrepeat sequence separated by three nucleotides located in the promoterof target genes. The DNA binding domain (DBD) in ERα and ER arevirtually identical, exhibiting 95% homology. The carboxy-terminusdomain contains the ligand binding domain (LBD), which carries outseveral essential functions. The LBD contains a region that forms alarge hydrophobic pocket where estrogenic compounds bind, as well asregions involved in ER dimerization. The LBD also contains a secondactivation function (AF-2) that interacts with coregulatory proteins.AF-2 is required for both estrogen activation and repression of genetranscription. The LBDs of ERα and ERβ are only about 55% homologous.The striking differences in the amino acid composition of the ERα andERβ LBDs may have evolved to create ERs that have distincttranscriptional roles. This would permit ERα and ERβ to regulate theactivity of different genes and to elicit different physiologicaleffects. This notion is supported by studies of ERα and ERβ knockoutmice. For example, the ERα knockout mice have primitive mammary anduterine development, whereas the ERβ knockout mice develop normalmammary glands and uterus. These observations demonstrate that only ERαis required for the development of these tissues. Furthermore, we havefound that ERα is more effective than ERβ at activating genes, whereasERβ is more effective than ERα at repressing gene transcription.

Mechanisms of action of estrogens: Estrogens can activate or repressgene transcription. There are two characterized pathways for activationof gene transcription, the classical ERE (estrogen response element)pathway and the AP-1 pathway. There are at least three essentialcomponents necessary for estrogens to regulate the transcription ofgenes: the ERs (ERα and/or ERβ), the promoter element in target genesand coregulatory proteins. The binding of estradiol to the ER leads to aconformational change, which results in several key steps that initiatetranscriptional pathways. First, the interaction of E₂ with ER leads tothe dissociation of chaperone proteins; this exposes the ER'sdimerization surface and DNA binding domain. Loss of the chaperoneproteins allows the ERs to dimerize and bind to an ERE in the promoterregion of a target gene.

Second, the binding of E₂ moves helix 12 of the ER's LED to create asurface that assembles the AF-2 function of the ER. The AF-2 consists ofa conserved hydrophobic pocket comprised of helices 3, 5 and 12 of theER, which together form a binding surface for the p160 class ofcoactivator proteins (coactivators), such as steroid receptorcoactivator-1 (SRC-1) or glucocorticoid receptor interacting protein 1(GRIP 1). Coactivators (also known as “coregulators”) contain severalrepeat amino acid motifs which project into hydrophobic cleft surroundedby the AF-2's helices. The coactivators possess histone acetylaseactivity. It is thought that gene activation occurs after the ERs andcoactivator proteins form a complex on the ERE that causes theacetylation of histone proteins bound to DNA. The acetylation ofhistones changes the chromatin structure so that the ER/coregulatorcomplex can form a bridge between the ERE and basal transcriptionalproteins that are assembled at the TATA box region of the target gene toinitiate gene transcription.

Effect of SERMs on the ERE pathway: Unlike estrogens, SERMs do notactivate the ERE pathway. Instead, the SERMs competitively block theeffects of estrogens on the ERE pathway. Like estrogens, SERMs bind toERα and ERβ with high affinity and cause the dissociation of chaperoneproteins, ER dimerization and binding of ERs to the ERE. Thus, theantagonist action of SERMs occurs at a step distal to the binding of theER to the promoter region. The molecular mechanism of the antagonistaction of the SERMs has been clarified by the crystallization of the ERαand ERβ LBDs. It is clear from the structure of the ER LBDs that E₂,tamoxifen and raloxifene bind to the same binding pocket. However,tamoxifen and raloxifene contain a bulky side-chain that is absent inE₂. The ER x-ray structures have revealed that the bulky side chain ofSERMs obstructs the movement of the LBD, which prevents the formation ofa functional AF-2 surface. Remarkably, when a SERM binds to ERα asequence in helix 12, interacts with the hydrophobic cleft of the AF-2surface to occlude the coactivator recognition site. Thus, unlikeestrogens, SERMs do not create a functional AF-2 surface; this preventsthe binding of coactivators. Because the coactivator proteins do notbind to the AF-2 surface in the presence of SERMs, the activationpathway is abruptly halted. Instead of recruiting coactivator, ERsliganded with SERMs recruit corepressors, such as N—CoR.

These studies demonstrated that the antagonist properties of SERMs aredue to at least three factors. First, SERMs bind to the same bindingpocket as estrogens and competitively block their binding to the ERs.Second, SERMs prevent ER from interacting with coactivator proteins thatare required for transcriptional activation of the ERE pathway. Third,SERMs recruit corepressors, which prevent transcriptional activation ofgenes. These actions of SERMs most likely explain how raloxifene andtamoxifen act as antagonists in breast cells to inhibit development ofbreast cancer.

SERMs are also more effective than E₂ at activating genes with an AP-1element. In fact, E₂ is an antagonist of SERM-mediated activation ofAP-1 elements. It has been postulated that SERMs exhibit agonisticactions in tissues, such as the bone and endometrium by activating theAP-1 pathway. Interestingly, SERMs are more potent at activating theAP-1 pathway in the presence of ERβ, which indicates that SERMs willtrigger the AP-1 pathway more efficiently in tissues that are rich inERβ. The role of the AP-1 pathway in estrogen-mediated breastcarcinogenesis is unclear, because estrogens are much weaker atactivating the AP-1 pathway compared to SERMs. However, it has beenproposed that the AP-1 pathway may be involved in resistance totamoxifen in breast tumors.

In accordance with aspects of the present invention, studies have beenperformed, which demonstrate that: ERβ is weaker than ERα at activatingERE-tkLuc; ERβ is more effective than ERα at repressing theTNF-RE-tkLuc; and that ERβ inhibits ERα-mediated transcriptionalactivation of ERE-tkLuc. Detailed experiments are discussed in theExamples section hereinafter.

The invention provides a plant extract composition that contains anextract of the taxonomic species Astragalus membranaceus Fisch. Bge.Var. mongolicus Bge. of the Leguminosae Family. An “extract” is acomposition of matter prepared by contacting an extraction medium(solvent) with plant matter under conditions suitable for drawing one ormore chemical compounds from the plant matter into the extractionmedium, forming an extraction solution. The extraction solution is thenseparated from the plant matter, and is optionally diluted or reduced,to form the extract.

The extract of the invention comprises phytochemicals obtained fromplant matter the plant species Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae Family. Plant matter is furtherdefined hereinafter.

The species Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. ofthe Leguminosae Family is also variously referred to as goat's horn,goat's thorn, green dragon, gum dragon, gum tragacanthae, gummitragacanthae, Huang Qi, locoweed, membranous milk vetch, milk vetch,Mongolian milk, Mongolian milk vetch, Syrian tragacanth, yellow vetch.Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae family is a herbaceous perennial shrub. It generally growsto a height of 40-80 cm. It has hairy stems with leaves made up of 12 to18 pairs of leaflets. One plant may have as many as 20 leaflet pairs.The plant bears small yellow flowers. Various cultivars are available,and may generally be obtained from commercial sources, such asnurseries.

The extraction medium is a suitable liquid solvent, e.g. ethyl acetate,water or ethanol. The extraction medium is in some cases ethyl acetate,water, ethanol or another relatively polar liquid solvent. In somecases, the extraction medium is either diluted or reduced. Theextraction medium may be fully reduced, whereby the extract takes theform of a residue (residual extract). Thus, the extract contains at aminimum one or more plant-derived compounds (phytochemicals), optionallydissolved in a solvent. A reduced or residual extract may bereconstituted by adding a suitable diluent, e.g. ethyl acetate, waterand/or ethanol, to form a reconstituted extract.

In some embodiments, compositions comprising plant extracts include pureextracts or partitioned extracts (including extracts in which one ormore estrogenically active compounds in the extract have been enriched)and combinations of such extracts with one or more additionalingredients. In some embodiments, the compositions include those in avariety of physical forms, including solid, semi-solid, liquid,colloidal, etc. Where the compositions are intended for pharmaceuticaluse, the additional ingredients are pharmaceutically acceptable. Wherethe compositions according to the invention are intended for use inassays or other uses that are not directed toward a living body, theadditional ingredient(s) may be either pharmaceutically acceptable ornot.

In some embodiments, a pure extract may be combined with one or moreorganic solvents. Such organic solvents may be of various polarities. Insome embodiments, suitable solvents include ethyl ethyl acetate,acetonitrile, hexanes, a (C₁-C₄) alcohol (e.g. methanol, ethanol,i-propanol, n-propanol, n-butanol, t-butanol, s-butanol, i-butanol,etc.), chloroform, acetone, cyclohexane, cycloheptane, petroleum ether,and other solvents, including those that are pharmaceutically acceptableand those that are generally regarded as safe (GRAS) for humanconsumption.

In some embodiments, the compositions comprise pure extracts orcombinations of extracts with one or more additional solvents. In someembodiments, the extract includes a partitioned or further purifiedextract. Partitioning or purification may be conducted using variousseparation techniques, including chromatography. In some embodiments,the extract is a purified or partitioned extract obtained by means ofanion exchange chromatography, cation exchange chromatography, reversephase chromatography, normal phase chromatography, affinitychromatography or exclusion chromatography, to further concentrateactive agents in the extract. In some embodiments, the purified orpartitioned extract is obtained via one or more steps of liquidchromatography, such as high performance liquid chromatography (HPLC).In some embodiments, high performance liquid chromatography ispreparative scale high performance liquid chromatography. In someembodiments, the HPLC is reverse phase or ion exchange chromatography.Other means of separation may also be used to purify or partition theextract, including separation in a separatory funnel or other bi- ormulti-phasic separatory mechanism. In some embodiments, the purified orpartitioned extract may be combined with one or more additional activeor inactive ingredients, such as solvents, diluents, etc. In someembodiments, suitable solvents may include ethyl acetate, acetonitrile,hexanes, a (C₁-C₄) alcohol (e.g. methanol, ethanol, i-propanol,n-propanol, n-butanol, t-butanol, s-butanol, i-butanol, etc.),chloroform, acetone, cyclohexane, cycloheptane, petroleum ether, andother solvents, including those that are pharmaceutically acceptable andthose that are generally regarded as safe (GRAS) for human consumption.

Suitable additional ingredients include solvents. Solvents may besubdivided into pharmaceutically acceptable and non-pharmaceuticallyacceptable solvents. In this context, it is to be understood that somepharmaceutically acceptable solvents include water for injection (WFI),which may be pH adjusted and/or buffered to a preselected pH or pHrange, e.g. from about 2 to about 8, more specifically from about 4.0 toabout 7.5, and more particularly from about 4.9 to about 7.2.

Pharmaceutically acceptable solvents may further comprise one or morepharmaceutically acceptable acids, bases, salts or other compounds, suchas carriers, excipients, etc. Pharmaceutically acceptable acids includeHCl, H₂SO₄H₃PO₄, benzoic acid, etc. Pharmaceutically acceptable basesinclude NaOH, KOH, NaHCO₃, etc. Pharmaceutically acceptable saltsinclude NaCl, NaBr, KCl, etc. Acids and bases may be added inappropriate proportions to buffer a pharmaceutically acceptable solutionat a particular, pre-selected pH, especially a pH in the range of about2-8, more especially in the range of about 5.0 to about 7.2

Plant extracts according to the present invention provide estrogenicactivation of genes under control of the estrogen response element(ERE). Accordingly, in some cells an inventive plant extract possessesestrogenic properties—i.e. contacting a cell comprising an ERE and an ER(ERα, ERβ or both) with an inventive plant extract gives rise tostimulation of a gene under control of the ERE. In an in vitro cellsystem, ERE-mediated activation by an inventive estrogenic plant extractleads to expression of a gene that is operatively linked to the ERE. Inparticular embodiments, estrogenic interaction of an ER with an ERElinked to the minimal thymidine kinase promoter and the luciferase genegives rise to enhanced luciferase expression. Thus, the plant extractsof the present invention may be used to identify ERα+ cell lines, ERβ+cell lines and/or ERα+/ERβ+ cell lines having an ERE-containing promoteroperatively linked to a reporter gene, such as luciferase. Plantextracts of the present invention may also be used as assay reagents,including standards, for identifying compounds having estrogenic effectsin ER+ cell lines.

In one such assay method, an inventive plant extract is first preparedat a known activity or concentration. Quantification of the inventiveplant extract is conveniently carried out by taring a container,measuring into the container a known volume of the plant extract,reducing the plant extract by evaporation or lyophilization to produce aresidue, and obtaining the mass of the container plus plant extract. Thedifference in mass between the container plus plant extract and the taremass is the dry mass of the plant extract. The ratio of dry mass ofplant extract per volume of plant extract is the concentration per unitvolume. The plant extract may be used in its initial form, using theresults of the foregoing quantitation method to specify itsconcentration. The residue can also be reconstituted by addition ofwater or another suitable solvent system to form a plant extractsolution of known concentration.

Once the concentration of plant extract is known, a standard curve isprepared. In general the ER+ cells are contacted with the plant extractand a signal relating to estrogenic activity is recorded. In particular,an ER+ cell has a reporter gene under the control of an ERE. This ER+cell is contacted with a plant extract of the invention, which givesrise to a reporter signal in proportion to the amount of plant extractadded. This step may be carried out with multiple samples at the sameplant extract concentration, at different plant extract concentrations,or both. As an example, nine samples may be tested: the first three at afirst concentration, the next three at a concentration that is a halflog greater than the first, and the next three at a concentration awhole log greater than first. The reporter signals are then observed andrecorded, and the resulting data points (plant extract concentrationversus reporter signal strength) are fitted to a standard curve by aconventional curve-fitting method (e.g. least squares).

To evaluate the estrogenic effect of a candidate compound, a candidatecompound is contacted with E+ cells having the reporter gene undercontrol of the ERE. The reporter gene signal is observed and compared tothe standard curve to quantitate the candidate compound's relativeestrogenic effect.

The ER+ cell line used in the foregoing method may be a cell line thatnaturally expresses ER, e.g. a human-derived ER+ breast cell carcinomacell line. In some embodiments, the ER+ tissue is an immortalized humancell line, e.g. an immortalized bone marrow or breast cell line.Exemplary cell lines include human monocyte, osteoblast, malignantbreast carcinoma and immortalized epithelial breast cell lines.Particular cell lines that may be mentioned include U937, U2OS,MDA-MB-435 and MCF-7 cell lines. Other ER+ cell lines, includingimmortalized cell lines, may also be used. Alternatively, the ER+ cellline may be a cell line that does not naturally express ER, such as abacterial cell line, that has been transformed with an ER expressionvector.

The ER+ cell line is transformed with a vector having a promotercontaining an ERE that controls a reporter gene. For example, the vectormay be a viral vector containing ERE, a minimal thymidine kinasepromoter (tk) and a luciferase gene (Luc). An exemplary ERE-tk-Lukconstruct is depicted in SEQ ID NO:1, where the ERE is represented bynucleotides 1, tk is represented by nucleotides nn-, and Luk isrepresented by nucleotides mm. The construct is transfected into thetarget cell by known methods and expression of the ER-ERE-tk-Luk systemis confirmed by e.g. performing the foregoing assay on putative ER+cells in the presence of known quantities of E₂. Other methods ofverifying successful transformation of ER+ cells include immunostainingwith known ER antibodies.

The ERE-containing promoter is a DNA containing an ERE sequence and apromoter sequence. The promoter sequence is an art-recognized promotersequence, such as the minimal thymidine kinase (tk) promoter sequence.(See SEQ ID NO:1, nucleotides nn). Other ERE-containing promoters arepossible and are within the scope of the instant invention. The ERE andpromoter sequence operate together to control expression of the reportergene. As described herein, the estrogenic compound (plant extract or E₂,for example) binds to the ER, giving rise to ER dimer and forming theAF-2 surface. The ER dimer then binds to the ERE, activating the geneunder control of the promoter. In some embodiments, the ERE is directlyupstream of (5′- to) the promoter, to which it is directly ligated. Asan example, the ERE-tk promoter construct is shown in SEQ ID NO: 1,nucleotides 1-nn-1.

The reporter gene is a gene which, when expressed, gives rise to adetectable signal. The luciferase gene is a suitable reporter genebecause it gives rise to the protein luciferase, which generates adetectable light signal in the presence of a single reagent, luciferin.In particular, the cDNA of the luciferase gene is expressed to producethe 62 kDa enzymatic protein, luciferase. The luciferase enzymecatalyzes the reaction of luciferin and ATP in the presence of Mg²⁺ andoxygen to form oxyluciferin, AMP, pyrophosphate (PPi) and emitted light.The emitted light is yellow-green (560 nm), and may easily be detectedusing a standard photometer. Because ATP, O₂ and Mg²⁺ are alreadypresent in cells, this reporter gene only requires addition of thereagent luciferin to produce a detectable signal, and is especiallywell-suited for use in assays of the present invention. Other reportergenes that may be mentioned as being available in the art includechloramphenicol transacetylase (CAT), neomycin phosphotransferase (neo)and beta-glucuronidase (GUS).

In some assay methods of the invention, it is useful to furthercharacterize the standard plant extract by comparison with one or moreestrogenic compounds, SERMs, etc. Such assay methods are performedessentially as described above, making the proper substitutions ofstandard estrogenic compound and/or SERMs for plant extract in theappropriate parts of the method.

Plant extracts according to the present invention also repress geneexpression by the TNF RE-mediated pathway. In some cases, plant extractsof the invention repress gene expression in vitro, especially in cellshaving a reporter gene (e.g. the luciferase gene, Luc) under control ofa TNF RE. In some cases, plant extracts of the invention repressexpression of TNF-α, which is a cytokine produced primarily by monocytesand macrophages. This cytokine is found in synovial cells andmacrophages in various tissues, and has been strongly implicated inrheumatoid arthritis (RA). TNF-α is also expressed in other inflammatorydiseases, and also as a response to endotoxins from bacteria. Asrepressors of TNF expression via the TNF RE repressor pathway, plantextracts of the invention are of interest in the treatment ofinflammatory disorders associated with elevated levels of TNF.

In some embodiments of the invention, a cell line is prepared, whichexpresses one or both of ERα and ERβ as well as a reporter gene undercontrol of TNF RE. The TNF RE is generally upstream of (5′- to) thereporter gene, and signal detection is carried out as previouslydescribed herein. The sequence of DNA having a reporter gene, in thiscase luciferase gene, under control of TNF RE is set forth in SEQ EDNO:2. Nucleotides 1-correspond to the TNF RE, while nucleotides nn-corresponds to the luciferase gene.

The foregoing cell TNF RE-containing cell system further contains one ormore copies of an ER gene—i.e. ERα, ERβ or both. The ER+ cell line usedin the foregoing method may be a cell line that naturally expresses ER,e.g. a human-derived ER+ breast cell carcinoma cell line. In someembodiments, the ER+ tissue is an immortalized human cell line, e.g. animmortalized bone marrow or breast cell line. Exemplary cell linesinclude human monocyte, osteoblast, malignant breast carcinoma andimmortalized epithelial breast cell lines. Particular cell lines thatmay be mentioned include U937, U205, MDA-MB-435 and MCF-7 cell lines.Other ER+ cell lines, including immortalized cell lines, may also beused. Alternatively, the ER+ cell line may be a cell line that does notnaturally express ER, such as a bacterial cell line, that has beentransformed with an ER expression vector.

In the presence of a predetermined amount of luciferin, and in theabsence of an estrogenic compound, e.g. E₂ or a plant extract of theinvention, the cell system emits a yellow light (560 nm) at anintensity, called the “control intensity” or the “baseline intensity”.Light emission at 560 nm is conveniently quantified in optical densityunits (O.D._(560nm)). Upon addition of an estrogenic compound, e.g. E₂or one of the inventive plant extracts, the intensity of 560 nm lightemissions is attenuated as compared to the control. Remarkably, in thepresence of a SERM, such as tamoxifen or raloxifene, luciferaseexpression increases and 560 nm light emission intensity also increases.Thus, plant extracts of the invention are capable of inducing anestrogenic TNF RE-controlled repression of gene expression.

The TNF RE-containing cell system can be used in an assay methodaccording to the invention. In the inventive assay methods, theattenuation of luciferase activity (i.e. decreased emission of 560 nmlight), correlates with increased estrogenic activity, whereasactivation of luciferase activity (i.e. increased emission at 560 nm),correlates with anti-estrogenic activity. Standard curves may beprepared using known quantities of the inventive plant extracts, asdescribed herein. Such standard curves may be further augmented by usingother known estrogenic or anti-estrogenic standards, such as E₂ or someother known estrogenic compound, and/or an anti-estrogenic SERM such astamoxifen or raloxifene.

Cells from the transformed E+ cell line are then exposed to a candidatecompound, the luciferase signal observed, and the signal compared to thepreviously prepared standard curve(s), as described herein. A compoundthat causes an increase of luciferase activity as compared to control(baseline), will be characterized as an anti-estrogenic SERM, whereas acompound that causes a decrease in luciferase activity versus controlwill be classified as estrogenic. The estrogenic or anti-estrogeniceffect can then be quantified by comparing the degree of luciferaseexpression decrease or increase against the decrease brought about bythe inventive plant extract, and optionally the respective signaldecrease or increase brought about by E₂, tamoxifen and/or raloxifene.

Plant extract compositions of the present invention also antagonize theinteraction of E₂-ER with ERE. In particular, it has been shown in thatextracts of Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. ofthe Leguminosae family antagonize the activation of ERE-tk-Luc by E₂ bydirectly interacting with ERβ and ERα. As antagonists of E₂-ERactivation of ERE-controlled genes, the inventive plant extractcompositions are considered to be similar in effect to tamoxifen,possessing prophylactic, palliative and/or anti-proliferative activityagainst breast cancer and uterine cancer.

The invention provides in vivo estrogenic methods of using the inventivecompositions. In general, in vivo methods comprise administering to asubject an amount of the plant extract sufficient to bring about anestrogenic effect in the subject. The in vivo methods will give rise toestrogenic ERE-controlled gene activation, TNF RE-controlled generepression (e.g. TNF-α repression), or both. Thus, the in vivo methodswill give rise to varied positive phenotypic effects in vivo.

The subject may be a mammal, such as a mouse, rat, rabbit, monkey,chimpanzee, dog, cat or a sheep, and is generally female. The subjectmay also be human, especially a human female. In some embodiments, thesubject is a post-menopausal or post-oophorectomic female, and is inneed of estrogenic therapy. In such case, the subject may be sufferingfrom climacteric symptoms, such as hot flashes, insomnia, vaginaldryness, decreased libido, urinary incontinence and depression. In othersuch cases, the subject may be susceptible to, or suffering from,osteoporosis. Suitable in vivo methods include treatment and/orprevention of medical indications that are responsive to estrogenreplacement therapy.

Administration of the compositions according to the present inventionwill be via a commonly used administrative route so long as one or moreof the plant extracts is available to target tissue via that route. Someadministrative routes that may be mentioned include: oral, nasal,buccal, rectal, vaginal and/or topical (dermal). Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra.

Treatment (and its grammatical variants—e.g. treat, to treat, treating,treated, etc.) of a disease, disorder, syndrome, condition or symptomincludes those steps that a clinician would take to identify a subjectto receive such treatment and to administer a composition of theinvention to the subject. Treatment thus includes diagnosis of adisease, syndrome, condition or symptom that is likely to beameliorated, palliated, improved, eliminated, cured by administering theestrogenic plant extract of the invention to the subject. Treatment alsoincludes the concomitant amelioration, palliation, improvement,elimination, or cure of the disease, disorder, syndrome, condition orsymptom. In some embodiments, treatment implies prevention or delay ofonset of a disease, disorder, syndrome, condition or symptom (i.e.prophylaxis), prevention or delay of progression of a disease, disorder,syndrome, condition or symptom, and/or reduction in severity of adisease, disorder, syndrome, condition or symptom. In the case ofneoplastic growth in particular, treatment includes palliation, as wellas the reversal, halting or delaying of neoplastic growth. In thisregard, treatment also includes remission, including complete andpartial remission. In the case of climacteric symptoms, treatmentincludes prevention and palliation of various symptoms.

Prevention (and its grammatical variants) of a disease, disorder,syndrome, condition or symptom includes identifying a subject at risk todevelop the disease, disorder, syndrome, condition or symptom, andadministering to that subject an amount of the inventive plant extractsufficient to be likely to obviate or delay the onset of said disease,disorder, syndrome, condition or symptom. In some cases, preventionincludes identifying a post-menopausal woman who the clinician believes,applying a competent standard of medical care, to be in need of hormonereplacement therapy, and administering a plant extract of the presentinvention to the woman, whereby one or more climacteric symptoms isblocked or delayed. In some embodiments, prevention of osteoporosisincludes identifying a post-menopausal woman who the clinician believes,applying a competent standard of medical care, to be at risk fordeveloping osteoporosis, and administering a plant extract of thepresent invention to the woman, whereby the onset of bone loss isblocked or delayed.

Palliation includes reduction in the severity, number and/or frequencyof occurrences of an a disease, disorder, syndrome, condition orsymptom. Palliation of climacteric symptoms includes reducing thefrequency and/or severity of hot flashes, insomnia, incontinence,depression, etc.

Treatment of osteoporosis includes identifying a person, such as apost-menopausal woman, at risk for bone loss, and administering a plantextract of the present invention to the woman, whereby bone loss isreduced in severity, delayed in onset, or prevented. In someembodiments, treatment of osteoporosis can also include addition of bonemass.

The invention further provides methods of making the inventive extractsof Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family. The invention specifically provides a method ofmaking an inventive estrogenic plant extract. The method includesobtaining a quantity of plant matter from a plant of the speciesAstragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family, optionally comminuting the plant matter, contactingsaid plant matter with an extraction medium, and separating the plantmatter from the extraction medium.

In some embodiments, the plant species are of the plant speciesAstragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family are various cultivars of Astragalus membranaceusFisch. Bge. Var. mongolicus Bge. of the Leguminosae Family.

Plant matter means any part or parts of at least one plant from thespecies Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family. Plant matter includes the whole plant or any part orparts of the plant, such as the root, bark, wood, leaves, flowers (orflower such as: sepals, petals, stamens, pistils, etc.), fruit, seedsand/or parts or mixtures of any of the foregoing. Plant matter may befresh cut, dried (including freeze dried), frozen, etc. Plant matter mayalso be whole or separated into smaller parts. For example, leaves maybe chopped, shredded or ground; roots may be chopped or ground; fruitmay be chopped, sliced or blended; seeds may be chopped or ground; stemsmay be shredded, chopped or ground. In particular embodiments of theinvention, the plant parts used are the leaves of Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae Family.

Plant extract compositions of the invention contain at least one extractof an Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae Family. An “extract” is a solution, concentrate or residuethat results when a plant part is contacted with an extraction solventunder conditions suitable for one or more compounds from the plant topartition from the plant matter into the extraction solvent; thesolution is then optionally reduced to form a concentrate or a residue.

Suitable extraction media for the present invention include water andethyl alcohol. Specifically, where water is the extraction solvent,purified water is suitable. Purified water includes distilled water,deionized water, water for injection, ultrafiltered water, and otherforms purified of water. Ethyl alcohol that is employed in someembodiments of the invention is grain ethanol, and in particularundenatured ethanol (e.g. pure grain ethanol, optionally containing somewater, e.g. up to about 10% water). In some embodiments, the extractionsolvent is water, ethanol, or a mixture thereof. A concentrate orresidue may be prepared by reducing (e.g. evaporating or lyophilizing)the extraction solution. Whether in the original extraction solvent,reduced concentrate, or residue form, each of these preparations isconsidered an “extract” for the purposes of the invention.

A method of producing the plant extract according to the inventionoptionally comprises first comminuting the plant matter in order toincrease its surface area to volume ratio and to concomitantly increaseefficiency of the extraction process. Methods of comminuting plantmatter include grinding, chopping, blending, shredding, pulverizing,triturating, etc.

The extraction medium (solvent) is then contacted with the plant matterunder conditions suitable for causing one or more phytochemicals, inparticular estrogenic phytochemicals, to partition from the plant matterinto the extraction medium. Such conditions include, in some cases,heating the extraction medium to a temperature above room temperature,agitation, contact time, etc. Exemplary temperatures for extraction arefrom about 50° C. to the boiling point of the extraction solvent. Wherewater is the extraction solvent, the extraction temperature is generallyfrom room temperature to about 100° C.; temperatures of from about 50°C. to about 80° C. are especially suitable, and temperatures of about75° C. are particularly suitable. In the case of ethanol as anextraction solvent, the extraction temperature is generally from aboutroom temperature to about 78.5° C.; temperatures of from about 50° C. toabout 78° C. are especially suitable and a temperature of about 75° C.is particularly suitable. The person of skill in the art will recognizethat the proper balance should be drawn between extraction efficiency onthe one hand and phytochemical compound stability on the other.

Once the extraction medium and the plant matter are combined, they areoptionally agitated to ensure efficient exchange of estrogenic compoundfrom the plant matter into the extraction medium, and are left incontact for a time sufficient to extract a useful amount ofphytochemical compound from the plant matter into the extraction medium.After such time has elapsed (e.g. from about 5 min to about 10 hr., moreparticularly from about 10 min. to about 5 hr., especially about 30 min.to about 2 hr.), the extraction medium containing the phytochemicalcompounds is separated from the plant matter. Such separation isaccomplished by an art-recognized method, e.g. by filtration, decanting,etc.

A composition according to the invention includes an inventive plantextract or a composition comprising an inventive plant extract of theinvention. In such embodiments, the inventive composition willoptionally contain one or more additional ingredients. Such additionalingredients may be inert or active. Inert ingredients include solvents,excipients and other carriers. Active ingredients include activepharmaceutical ingredients (APIs), including those that exhibitsynergistic activity in combination with the inventive plant extract.

EXAMPLES

The invention may be more fully appreciated with reference to thefollowing illustrative and non-limiting examples.

Example 1

ERβ is weaker than ERα at activating ERE-tkLuc: The effects of E₂ ontranscriptional activation were examined by transfecting a plasmidcontaining a classical ERE upstream of the minimal thymidine kinase (tk)promoter linked to the luciferase reporter cDNA and an expression vectorfor ERα or ERβ. E₂ produced a 10-fold greater activation of the ERE inthe presence of ERα compared to ERβ in human monocytic U937 cells, butthe EC50 values were similar.

Example 2

ERβ is more effective than ERα at repressing the TNF-RE-tkLuc: Theeffects of effects of E₂ on ERα and ERβ-mediated transcriptionalrepression were then compared using the −125 to −82 region of the TNF-αpromoter, known as the tumor necrosis factor-response element (TNF-RE).TNF-α produced a 5-10-fold activation of 3 copies of the TNF-RE (−125 to−82) upstream of the tk promoter (TNF-RE tkLuc). E₂ repressed TNF-αactivation of TNF-RE tkLuc by 60-80% in the presence of ERα and ERβ.However, ERβ was approximately 20 times more effective than ERα atrepression (IC₅₀ of 241 pM for ERα versus 15 pM for and ERβ,respectively). It was also found that ERβ is more effective than ERα atrepressing the native −1044 to +93 TNF-α promoter. Thus, ERα is muchmore effective than ERβ at transcriptional activation, whereas ERβ ismore effective than ERα at transcriptional repression. In contrast toE₂, the antiestrogens, tamoxifen, raloxifene and ICI 182, 780 produced a2-fold activation of TNF-RE tkLuc. Furthermore, these antiestrogensabolished the repression induced by E₂.

Example 3

ERβ inhibits ERα-mediated transcriptional activation of ERE-tkLuc:Surprisingly, when ERα or ERβ were coexpressed in U937 cells, theactivation by ERα is markedly inhibited (FIG. 1). These data show thatERβ exerts a repressive effect on ERα activation of ERE-tkLuc. Similarresults were observed in the breast cancer cell line, MDA-MB-435 (FIG.2). Other investigators have found a similar repressive effect of ERβ onERα transactivation in different cell types. These studies indicate thatthe different activation of ERα and ERβ on ERE-tkLuc and the repressiveeffect of ERβ on ERα-mediated-transcription are not cell-type specificand results from intrinsic properties of the ERs. The repression of ERαby ERβ requires the formation of an ERα/ERβ heterodimer, becausemutations in helix 11 of ERβ that prevent dimerization inhibit itsrepression activity (data not shown).

Example 4

Materials and Methods: Reagents. Phenol red-free Dulbecco's modifiedEagle's/F-12 Coon's modification medium was obtained from Sigma.Biobrene was purchased from Applied Biosystems. The U937 cell line wasobtained from American Type Culture Collection. Human recombinant TNF-αwas obtained from R & D Systems.

Plasmid Construction. A PstI to AhaII. fragment (−1044 to +93) from thehuman TNF-α gene, pLT, was cloned upstream of the luciferase cDNA. The5′ deletions were constructed by using unique restriction sites, ApaIfor the −125 deletion, and StyI for the −82 deletion. Three copies ofthe human TNF-α promoter fragment from −125 to −82 [TNF-responsiveelement (TNF-RE)] or one copy of the ERE from the frog vitellogenin A2gene (vitA2-ERE) were ligated upstream of −32 to +45 herpes simplexthymidine kinase (TK) promoter linked to luciferase (TNF-RE tkLuc, andERE TKLuc, respectively). ERβ mutants were created with QuikChangesite-directed mutagenesis kits (Stratagene), by using oligonucleotidescontaining the mutation. The mutants were sequenced with Sequenase kits(Amersham Pharmacia) to verify the presence of the mutation.

Cell Culture, Transfection, and Luciferase Assays—U937 (human monocyte),U205 (human osteosarcoma), MDA-MB-435 (human metastatic breast cancer),and MCF-7 (human breast cancer) cells were obtained from the cellculture facility at the University of California, San Francisco. U937cells were maintained as described previously, whereas U205, MDA-MB-435,and MCF-7 cells were maintained and subcultured in phenol red-freeDulbecco's modified Eagle's medium/F-12 media containing 5% fetal bovineserum, 2 mM glutamine, 50 units/ml penicillin, and 50 μg/mlstreptomycin. For experiments, cells were collected, transferred to acuvette, and then electroporated with a Bio-Rad gene pulser as describedpreviously using 3 μg of reporter plasmid and 1 μg of ERα or ERβexpression vectors. After electroporation, the cells were resuspended inmedia and plated at 1 ml/dish in 12-well multiplates. The cells weretreated with E₂, genistein, daidzein, or biochanin A (Sigma-Aldrich) 3hr prior to exposure to 5 ng/ml TNF-α (R & D Systems) for 24 hr at 37°C. Cells were solubilized with 200 μL of 1× lysis buffer, and luciferaseactivity was determined using a commercially available kit (Promega).The concentration of hormone required to produce a half-maximalinduction (EC₅₀) or inhibition (IC₅₀) of luciferase activity wascalculated with the Prism curve-fitting program (Graph Pad Software,version 2.0b). For proliferation studies, parental MCF-7 cells weresubcloned at 1 cell/well in the presence of 0.1 nM E₂, and the fastestgrowing clone was selected for experiments. These cells expressedexclusively ERα as determined by reverse transcription polymerase chainreaction (RT-PCR). The cells were plated in duplicate at a density of25,000 cells/35-mm plate in tissue culture medium containing 3% strippedfetal bovine serum. One day after plating they were treated withincreasing concentrations of E₂ or genistein. The medium was changedevery other day, and E2 or genistein was added to the medium. After 8days the cells were counted with a Coulter counter. All experimentspresented in the figures were performed at least three times, and thedata were similar between experiments.

Preparation of Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge.of the Leguminosae family of the Liliaceae family: Samples of Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familywere ground to fine powder using a commercial electric herb grinder; 5grams were weighed and extracted in a) 50 ml of 100% EtOH or b) 50 ml ofdistilled H₂O was simmered at 75° Celsius. for 45 minutes. The extracts(a and b) were than decanted and only the soluble material was used.

Results: Selective estrogen receptor modulating activity in U2OS Bonecells was measured using luciferase assays. U2OS osteosarcoma cells werecotransfected with a classic ERE upstream of a minimal thymidine kinase(tk) promoter (ERE-tk-Luc) and expression vectors for human ERα or ERβ.Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae family activation of ERE-tk-Luc with ERβ, and ERα. ERβproduced a 4.67-fold activation of ERE-tk-Luc with 1 μL/ml Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familyand a 4.03-fold activation of ERE-tk-Luc with 1 μL/ml on ERα. Theseresults indicate that Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae family activates ERE-tk-Luc bydirectly interacting with ERβ.

To investigate the effects of Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae family on transcriptional repression,the −125 to −82 region of the TNF-α promoter (TNF-α-responsive element,(TNF-RE)) was used because this region mediates TNF-α activation and E₂repression. E₂ produced a profound repression of TNF-α activation of theTNF-RE upstream of a minimal tk promoter (TNF-RE tkLuc) with eithertransfected ERα or ERβ in U2OS cells. E₂ can abolish TNF-α activity onERβ (100% repression) but not on ERα (73.3% repression). Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familyproduced a large repression of TNF-α activation of TNF-RE in thepresence of ERβ (109.6%) and ERα (102.8%). These results indicate thatAstragalus membranaceus Fisch. Bge. Var. mongolicus Bge. of theLeguminosae family represses TNF-α activation through TNF RE-tk-Luc bydirectly interacting with ERβ and ERα.

In these experiments, the lowest dose of a composition comprisingextract of Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. ofthe Leguminosae family that is effective for estrogenic activity is 1.2μg. However, it is to be expected that in other cell systems this numbermay fluctuate.

Example 5 Open Label, Increasing Dose, Dosing Study

In order to assess the safety and maximum tolerated dose (MTD) of anextract of Astragalus membranaceus Fisch. Bge. Var. mongolicus Bge. ofthe Leguminosae Family (Study Drug), the following protocol is carriedout. Study Drug comprises 1 mg (week 1), 10 mg (week 2), 100 mg (week 3)or 1000 mg (week 4) of extract of Astragalus membranaceus Fisch. Bge.Var. mongolicus Bge. of the Leguminosae Family in a suitably sizedgelatin capsules. (Hereinafter the extract of Astragalus membranaceusFisch. Bge. Var. mongolicus Bge. of the Leguminosae Family may bereferred to as “Study Drug”). The dose may be split between two or moregelatin capsules if necessary. Normal, healthy volunteers of age 18 to60 are administered 1 mg per day of Study Drug for week 1, 10 mg per dayof Study Drug for week 2, 100 mg per day of study drug for week 3 and1000 mg per day of Study Drug for week 4. Subjects are monitored forappearance of any adverse events. At any time, if a subject appears tonot tolerate the current dose, the attending medical staff will notesuch intolerance. The maximum tolerated dose will be considered thehighest dose at which each of the subjects tolerates the dose, or, if nosubject experiences intolerance, 1000 mg of the Study Drug per day.

Example 6 Double Blind Efficacy Study

In order to demonstrate efficacy of the Study Drug for the treatment ofestrogenic disease states, the following double blind study isperformed.

Objective:

To determine optimal dose and the safety and efficacy of an ERβselective Chinese herbal extract (Study Drug) for treatment of hotflushes (also known as hot flashes).

Methods:

A multicenter, randomized, blinded, phase II, placebo-controlled trialin 100-300 generally healthy postmenopausal women aged 40-60 yearsreporting at least 7 moderate to severe hot flushes per day or 50 perweek. Women are randomized to 5 g (SG5) or 10 g (SG10) per day of StudyDrug or identical placebo (PG) for 12 weeks. Hot flush frequency andseverity are recorded in a daily diary.

Results:

Participants are characterized by mean age and race. Participantsreceiving both Study Drug and placebo are also characterized by percentdecrease (±S.D., and p value) in hot flush frequency after 12 weeks oftreatment. Endometrial thickness is evaluated for each participant andeach group (overall, PG, SG5, SG10). Adverse events are also evaluatedfor each participant and each group (overall, PG, SG5, SG10).

Conclusions:

Evaluation is based upon the reduction in frequency and severity of hotflushes in healthy postmenopausal women as well as dose titrationeffects.

Methods

Design and Setting:

This is a multi-center, randomized, blinded, placebo-controlled trialdesigned to determine whether the Study Drug is safe and effective inreducing the frequency and severity of hot flushes. The trial iscoordinated through an independent third party (Coordinating Center) andparticipants are recruited at multiple clinical sites.

Participants:

Eligible participants are generally healthy postmenopausal women 40 to60 years old who reported at least 7 moderate to severe hot flushes perday or 50 per week. Women who are excluded: those with a history ofbreast, uterine or ovarian cancer; melanoma; venous thromboembolism;cardiovascular disease, or severe food or medicine allergies. Alsoexcluded are women reporting active liver or gallbladder disease;abnormal uterine bleeding; pregnancy or lactation, and those with anabnormal mammogram, breast examination, Pap smear or pelvic examinationsuggestive of cancer. Women with endometrial thickness exceeding 5 mmmeasured by transvaginal ultrasound and those using medications known orsuspected to affect hot flushes (estrogens, tamoxifen, raloxifene,progestins, selective serotonin reuptake inhibitors or gabapentin) arealso excluded.

At screening, placebo medication and diaries to record hot flushes,bleeding and medication adherence are provided for a 1-week run-inperiod. Participants who correctly complete their diaries, take at least80% of the placebo medication, and remain eligible after screeningphysical, radiological, and laboratory exams are randomized.

Drug safety is evaluated by a Data Safety and Monitoring Board.

Data Collection:

Data are collected, cleaned and analyzed by the Coordinating Center.

Randomization:

Randomization is stratified by time since last menstrual period (≦24months vs. >24 months) and by clinical site; within strata, treatment israndomly assigned in randomly permuted blocks of 3 to 6 in a 1:1:1ratio. A research pharmacist at the Coordinating Center receives thestudy medication from Bionovo, Inc. (Emeryville, Calif.), applies labelswith treatment identification numbers generated by the CoordinatingCenter statistician, and ships study medication to each clinical site.Study medication is allocated to eligible participants sequentiallyaccording to the randomization scheme.

Study Medications and Blinding:

Study Drug is a filtered, dried extract of herb as described herein.Carmel coloring and food dyes approved by the US Food and DrugAdministration are added to the dry powder to reach a uniform color, andflavorings and sweeteners are added to mask the taste of the herbs.Similar coloring and taste excipients are added to inert solid diluentto produce a placebo powder with the same look, taste and granularity asthe active medication.

Participants receive placebo or one of the two doses of Study Drugpackaged as a powder and are instructed to dissolve the contents of thepacket in at least 3 ounces of non-citrus fluid and drink the beveragetwice daily. All investigators, study staff, laboratory personnel andparticipants are blinded to study medication status.

Measurements:

At baseline, participants complete questionnaires regardingdemographics, medical, history, medications, quality of life, menopausalsymptoms, insomnia (Insomnia Severity Index) and sexual function (FemaleSexual Function Index). All participants receive a physical examination,including blood pressure and heart rate, a breast and pelvic exam, and,in women without a hysterectomy, a transvaginal ultrasound to measureendometrial double wall thickness. To evaluate safety, serum hematology,creatinine and urea nitrogen, liver function, and a urine analysis areall performed for each patient. All baseline measures are repeated after12 weeks of treatment or at the final study visit.

Hot flush frequency and severity are recorded on a diary modeled after adiary widely used in prior studies. The 7-day diary is completed priorto randomization and during weeks 4 and 12 on study medication. For eachhot flush, severity is rated as 1 (mild), 2 (moderate) or 3 (severe). Ahot flush score is calculated by adding the severity rating for each hotflush and dividing by the number of hot flushes.

While on study medication, participants are contacted (by phone or inthe clinic) at 2 and 8 weeks, and have a clinic visit at 4 weeks tomonitor adherence and adverse events. Medication packets are counted toassess adherence; and adverse events are recorded.

Four weeks after discontinuing study medication, each participant iscontacted by phone to ascertain information on adverse events.Self-reported adverse events are classified using the Medical Dictionaryfor Regulatory Activities (MedDRA) system.

Diagnostic endometrial biopsies are performed during the study if aparticipant reports vaginal spotting or bleeding, or if the finalendometrial wall thickness measured by transvaginal sonography is over 5mm or has increased 2 mm or more from baseline. Two blinded pathologistsevaluate biopsy specimens, if any, independently. If the pathologistsdisagree regarding histology, another third blinded pathologist reviewsthe slide and makes the final diagnosis.

Statistical Analysis:

A sample of 180 participants is estimated to provide 80% power to detecta between-group difference of 20 percentage points in the percent changein hot flush frequency from baseline to 12 weeks.

All analyses are by intention to treat, according to randomizedassignment, without regard to adherence and without imputing or carryingforward missing values. No adjustment is made for multiple testing.Baseline characteristics of the participants are compared using linearor logistic regression or proportional odds models controlling forclinical center and years since menopause.

Primary analyses compare changes from baseline to 4 and 12 weeks infrequency of hot flushes and hot flush score between each of the StudyDrug groups (SG5 and SG10) and placebo (PG). Because the outcomes areright-skewed, repeated-measures log-link Poisson generalized linearmodels with terms for time (4 or 12 weeks vs. baseline), treatment, anda time-by-treatment interaction, as well as clinical center and yearssince menopause are used. Primary analyses of secondary outcomes(quality of life, sexual function and insomnia scores) use analogousmethods.

In secondary analyses, ANCOVA is used, controlling for site and timesince menopause to compare rank transformed percent change in number ofhot flushes between the treated and placebo groups. Logistic regressionmodels adjusted for clinical site and years since menopause are used tocompare the proportions in each treatment group with a reduction infrequency of hot flushes of 50% or greater from baseline to 12 weeks.

The frequency of adverse events that occurs in more than 2% of any ofthe treatment groups is compared between treatment groups usingchi-square and exact methods when appropriate, stratified by clinicalcenter and years since menopause.

In pre-specified exploratory analyses, interaction terms are used todetermine differences in the treatment effect (percent change in hotflushes at 12 weeks) in subgroups including age (45-50; 50-55; 55-60years) ethnicity (white, other), years since menopause (less than 2years; 2 years or more), bilateral oophorectomy (yes; no), history ofestrogen use (yes; no), smoking (current; former or never), currentalcohol use (yes, no), body mass index (tertiles), baseline serumestradiol level (5 pg/ml or less; greater than 5 pg/ml), and baselinefrequency of hot flushes (tertiles).

Results

Results include number of eligible women who are randomized; number ofwomen in each group (PG, SG5, SG10); number of participants who completethe study overall and in each group and strata; number of participantsoverall and in each group who took all the assigned medication; numberof white and non-white participants overall and in each group; baselinemedian and mean daily frequency of hot flushes (±S.D., p); median andmean daily hot flush score (±S.D., p); median and mean change in hotflush frequency (±S.D., p) and median and mean hot flush score (±S.D.,p) at each evaluation interval.

The effects of treatment with Study Drug on measures of quality of life,sleep quality and sexual function as compared to placebo are alsoevaluated.

The number of participants receiving transvaginal ultrasound at baselineand the end of the study is also noted. The number of participantsreceiving endometrial ultrasound at the end of the trial is also noted.Mean endometrial thickness (±S.D.) at baseline and at 12 weeks ismeasured. Where deemed necessary, endometrial biopsy is also performed.The number of participants reporting vaginal bleeding or spotting isalso noted; and endometrial biopsy is in as many of these participantsas grant consent. The biopsies are evaluated for evidence of endometrialhyperplasia and cancer.

Any serious adverse events during the trial are also noted.

Discussion

It is considered that treatment with the Study Drug will decrease thefrequency and severity of hot flushes in healthy postmenopausal womenwith moderate to severe symptoms. The results of this study may be usedto advance the Study Drug on to further clinical trials, in which thesame or higher doses of Study Drug may be tested.

It is also considered that, as the Study Drug is a selective ERβagonist, adverse events associated with estrogen replacement therapy,such as uterine hyperplasia and cancer, should not be observed for theStudy Drug.

While estradiol is an effective treatment for menopausal hot flushes,the currently approved selective estrogen receptor modulators (SERMS)tamoxifen and raloxifene increase the incidence of menopausal hotflashes. Since neither estradiol nor the SERMs are estrogen receptorsubtype selective, it is unclear which estrogen receptor, ERα or ERβmediates these effects. It has been shown that activation of ERα byestrogen in human breast cancer cells results in proliferation and tumorformation, while activation of ERβ results in growth inhibition and notumor formation. This study is designed to provide data to demonstratethat hot flushes may be relieved by the Study Drug. This study isfurther designed to provide preliminary data regarding adverse eventsthat may be associated with the Study Drug.

Conclusion:

Treatment with the Study Drug is expected to reduce the frequency andseverity of hot flushes in healthy postmenopausal women; and the higherdose of the Study Drug is expected to be more effective than the lowerdose. This study is furthermore expected to provide further confirmationthat the ERβ pathway may play a role in the treatment of hot flushes.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of modulating the expression of a geneunder control of an estrogen response element, wherein modulation of thegene under control of the estrogen response element treats at least oneclimacteric symptom, comprising administering to a female human anamount of a composition consisting of an extract of Astragalusmembranaceus Fisch. Bge. Var. mongolicus Bge. of the Leguminosae familysufficient to modulate said gene under control of the estrogen responseelement and at least one active pharmaceutical ingredient selected fromraloxifene and tamoxifen, wherein the extract includes one or morecompounds capable of binding at least one of estrogen receptors ERα andERβ in cells growing in vitro or in vivo.
 2. The method of claim 1,wherein the climacteric symptom is one or more of hot flashes, insomnia,vaginal dryness, decreased libido or urinary incontinence.
 3. The methodof claim 1, wherein the climacteric symptom is hot flashes.
 4. Themethod of claim 1, wherein the amount of the extract included in thecomposition is in a range from 1 mg to 10,000 mg.
 5. A method ofmodulating the expression of a gene under control of an estrogenresponse element, wherein modulation of the gene under control of theestrogen response element treats at least one climacteric symptom,comprising administering to a female human an amount of a compositionconsisting of an extract of Astragalus membranaceus Fisch. Bge. Var.mongolicus Bge. of the Leguminosae family sufficient to modulate saidgene under control of the estrogen response element and at least oneactive pharmaceutical ingredient selected from raloxifene and tamoxifen,wherein the composition comprises at least two separate units: a firstunit comprising the extract and a second unit comprising the at leastone active ingredient.